CYP Inhibition, LC-MS/MS Detection (IC50 Determination)
Assay System:
- Human liver microsomes, pooled from 50 donors.
- Inhibition potential of test compound measured upon incubation with human liver microsomes at different compound concentrations. Reversible inhibition is determined by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
Assay Conditions:
| Test Compound Concentration | Eight concentrations ranging from 0.05 µM to 100 µM |
| Percent Organic Solvent in Incubation | 2% acetonitrile |
| CYP Isoforms Available for Testing | CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 |
| Incubation Time | 10-20 minutes (depending on CYP isoform) |
| pH | 7.4 |
| Temperature | 37 °C |
| Analysis | LC-MS/MS |
| Number of Repeats | 2 |
Deliverable: IC50 of test compound and positive control compound.
Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.
| CYP Isoform | Probe Substrate | Monitored Metabolite | Positive Control Inhibitor |
|---|---|---|---|
| 1A2 | Phenacetin | Acetaminophen | Furafylline |
| 2B6 | Bupropion | OH Bupropion | Ticlopidine HCl |
| 2C8 | Amodiaquine | Desethylamodiaquine | Montelukast |
| 2C9 | Tolbutamide | 4’-OH Tolbutamide | Sulfaphenazole |
| 2C19 | S-Mephenytoin | 4’-OH Mephenytoin | (+)-N-3-Benzylnirvanol |
| 2D6 | Dextromethorphan | Dextrorphan-D-Tartrate | Quinidine |
| 3A4 | Testosterone | 6β-OH Testosterone | Ketoconazole |
| 3A4 | Midazolam | 1’-OH Midazolam | Ketoconazole |